Definition of optimal substrate recognition motifs of Ca2+-calmodulin-dependent protein kinases IV and II reveals shared and distinctive features.
The substrate recognition determinants of Ca2+-calmodulin-dependent protein kinase (CaMK) IV and CaMKIIalpha were investigated using peptide substrates modeled on the amino acid sequence encompassing Ser-9 of synapsin I. For both kinases, hydrophobic residues (Leu or Phe) at the -5 position, are well tolerated, whereas non-hydrophobic residues (Arg, Ala, or Asp) decrease Vmax/Km by 55- to >4000-fold. At the -3 position, substitution of Ala for Arg leads to decreases of 99- and 343- fold in Vmax/Km for CaMKIV and CaMKIIalpha, respectively. For both kinases, the nature of the residues occupying the -4, -1, and + 4 positions exerts relatively little influence on phosphorylation kinetics. CaMKIV and CaMKIIalpha respond differently to substitutions at the -2 and +1 positions. Substitution of Arg at the -2 position with non-basic residues (Gln or Ala) leads to 6-fold decreases in Vmax/Km for CaMKIV, but 17-28-fold increases for CaMKIIalpha. Additionally, peptides containing Leu, Asp, or Ala at the +1 position are phosphorylated with similar efficiencies by CaMKIV, whereas the Leu-substituted peptide is preferred by CaMKIIalpha (by a factor of 5.8-9.7-fold). Thus, CaMKIV and CaMKIIalpha preferentially phosphorylate substrates with the motifs: Hyd-X-Arg-X-X-Ser*/Thr*, and Hyd-X-Arg-NB-X-Ser*/Thr*-Hyd, respectively, where Hyd represents a hydrophobic, X any, and NB a non-basic amino acid residue. The different specificities of the two kinases may contribute to their targeting to distinct physiological substrates during Ca2+-dependent cellular events.
White
R R
RR
Department of Pharmacology and Toxicology, State University of New York, Buffalo, New York 14214, USA.
Kwon
Y G
YG
Taing
M
M
Lawrence
D S
DS
Edelman
A M
AM
D000595:Amino Acid Sequence; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D007700:Kinetics; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity
D010455:Peptides; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; D017871:Calcium-Calmodulin-Dependent Protein Kinases